Mitotic spindle assembly requires the rapid dissolution of the interphase microtubule (MT) cytoskeleton, including disassembly of both dynamic and non-dynamic MTs. While the turnover of microtubules during interphase and mitosis has been well characterized, we know relatively little about how the interphase MT array is removed to make way for assembly of a bipolar spindle. Interphase and mitotic MT arrays exist at near steady states, while the dissolution of the interphase MT array represents a transition between these two steady states. How the entire MT array transitions between self-organized states has not been explored, yet is critical to understanding events where the MT array shifts between arrays of different patterns (e.g. mitosis, and neuronal process formation). Whatever the mechanism underlying MT array transition from G2 to prometaphase of mitosis, it is surprisingly uninhibited by Taxol, a MT stabilizing drug used to treat several human cancers. Here we propose a combination of computer simulation and cell-based experiments to define how the interphase MT array is removed just prior to mitosis and to establish when various steps occur relative to the level of active CDK/cyclin B. Our experiments take advantage of our recent discovery that we can significantly increase the number of cells for study by blocking cells in G2 of the cell cycle with a CDK1 inhibitor (RO3306) and then follow mitotic entry within 30 min of drug washout. We propose 3 aims to define MT turnover and dissolution at mitotic entry. In Aim 1 we will determine the pathway of dynamic MT reorganization by combining computational simulations of the entire MT array's length distribution, MT number and total polymer with experimental measures of these same features. In Aim 2 we define when stable, non-dynamic MTs are depolymerized and whether they form the MT bundles present at prophase. In Aim 3 we explore mechanisms underlying MT bundle disassembly, using both MT marking experiments to localize depolymerization to MT plus and/or minus ends, and by probing the roles of MT motors, MT depolymerizing kinesins and MT severing proteins in dissolution of MT bundles.